Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions (AU)

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Intracellular calcium homeostasis in Leishmania mexicana. Identification and characterization of a plasma membrane calmodulin-dependent Ca(2+)-ATPase

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...

Actividad proteín-quinasa endógena y fosforilación de protaínas intracelulares en cáncer de mama: relación con elementos hsitológicos de pronóstico / Endogenous protein-kinase activity and intracellular protein phosphorylation in breast cancer: relationship with histological elements of prognostic

Existen evidencias previas que demuestran un aumento de la actividad global de proteín-quinasa en tejido mamario canceroso, lo que se traduce en patrones de fosforilación proteica alterados, tanto para proteínas de membrana (plasmática y nuclear) como citoplásmicas. La fosforilación proteica es un importante mecanismo de regulación de ciclo celular, de modo que el hallazgo de fosforilaciónes anormales dentro de las células patológicas puede constituir un indicio de la alteración que induce la neoplasia, y en la práctica podría relacionarse con la evolución del tumor. Con el objeto de cuantificar los niveles de fosforilación endógena de proteínas y de demostrar que los patrones de fosforilación en las células neoplásicas son diferentes de los de la células no neoplásicoas, se realizó un estudio en homegenizado de tejido mamario obtenido de 18 pacientes, en cada una de las cuales se extrajó una muestra de tejido canceroso y otra de parénquima mamario normal. En ellas se determinó la actividad PK endógena con técnica de Roskoski modificada y se determinó los patrones de fosforilación mediante análisis electroforético y posterior exposición en placas de autorradiografía.En 17 de los 18 casos, se detectó una diferencia entre los tejidos normales y los neoplásicos reflejado en los patrones de fosforilación respectivos. Las diferencias más notables se encontraron en la fosforilación endógena de la proteína de 59,5 kd que se fosforila en el 88,9 por ciento de los cánceres y en el 16,7 por ciento de los tejidos normales; la proteína de 13 kd que se fosforila en el 72,2 por ciento de los cánceres y en el 22,2 por ciento de los tejidos normales y en la proteína de 150 kd que se fosforila en el 94,4 por ciento de los cánceres y en el 16,7 por ciento de los tejidos normales. Las diferencias observadas demuestran que existe una notable alteración en los mecanismos de regularización de la fosforilación proteíca dentro de la célula de cáncer de mama